The goal of gene therapy is to deliver genetic material of therapeutic value to a target tissue in a safe and efficient manner. Safety is often related to how much damage is done to the normal tissues of the patient during treatment. Efficiency can be looked at as a ratio of desired result, e.g. reduction of tumor load, to acceptable dosage level, where the parameters which contribute to making a dosage level “acceptable” can include issues of injection volume, frequency, etc. Therefore, any improvements that result in an increase in the selectivity and efficiency of gene therapy are clearly desirable.
Viral vectors derived from adenoviruses, have been the most studied delivery agents for this type of therapy (Jolly, D. (1994) Cancer Gene Therapy 1:51-64). Replication-defective vectors are limited in their usefulness due to their ability to only kill tumor cells that have been directly infected with virus. Viral vectors developed from oncolytic (i.e. replication-competent) viruses are more attractive choices for cancer therapeutics because they not only selectively target tumor cells, but they can, through replication within the infected tumor cells, amplify and spread the input dose of infective virus throughout the tumor cell mass.
The potential use of vectors derived from oncolytic viruses, such as adenovirus, in gene therapy can be further increased by “arming” the viruses with therapeutic transgenes, i.e. engineering them to contain therapeutic proteins or other molecules whose in vivo expression can impact tumor survival (Hermiston, T. (2000) J. Clin. Inv. 105:1169-1172). The combination of viral replication within tumor cells and the activity of the therapeutic molecule expressed within the cells can provide a synergistic assault on a tumor.
The incorporation of therapeutic transgenes into an oncolytic virus is a complex process. The insertion event needs to occur in a site that maintains the replication competence of the viral agent, which is complicated as viruses maximize their coding capacity by generating highly complex transcription units controlled by multiple promoters and alternative splicing (Akusjarui and Stevenin (2003 Curr. Top. Microbiol. Immunol. 272:253-286). Consequently, the choice of insertion sites for therapeutic genes has been limited primarily to regions known to be non-essential for viral DNA replication in vitro (Hawkins et al. (2001) Gene Ther. 8:1123-1131; Kurihara et al. (2002) J. Clin. Invest 106:763-771) or by the replacement of a deleted region of the viral genome to create the oncolytic virus (Freytag et al. (1998) Human Gene Ther. 9:1323-1333; Lee et al. (2001) Cancer Gene Ther. 8:397-404). While these approaches allow for therapeutic gene insertion and expression, they are dependent upon a high level of understanding of the viral biology (i.e. sites non-essential for viral replication), a known viral genome sequence (for use in genetic engineering or utilization of endogenous restriction enzyme sites) and the presence of molecular biology systems for genomic manipulations that may not be currently available for non-Ad5-based systems.
In view of the above, there is a need for a method for generating replication competent viruses which contain genetic elements, e.g., a gene which encodes a therapeutic protein or RNA, positioned within the viral genome such that expression of the genetic element occurs. Of particular utility would be a method that identifies functional insertion sites within replication competent viruses whose genomic structure has not yet been elucidated.